The sodium glucose symporter (a well characterized apical membrane transporter in the proximal tubular epithelium of the nephron) will be used as a model system in which to study the generation and maintenance of epithelial cell polarity. Specifically, the questions relate to how the symporter is processed intracellularly and routed to the apical as opposed to the basolateral membrane; what domains in the protein are necessary for membrane insertion, translocation and transport function; how the subunits within the membrane form a functional symporter; what role (if any) the cellular cytoskeleton, other membrane proteins and recognition particles play in these processes. The bulk of Phase I of the award will be spent isolating, purifying, reconstituting and cloning the symporter (in addition to a rigorous didactic program consisting of coursework, tutorials and seminars) in order to begin to address these questions using site-directed mutagenesis, expression of the mutated cDNA and antipeptide antibodies in Phase II of the award. Dr. Gunter Blobel's laboratory at the Rockefeller University, where the work will be done, not only has considerable expertise in these technologies but is also considered a leader in the field of intracellular protein topogenesis and membrane-protein interactions. These approaches should yield valuable insights into transport protein function and transport disorders in the kidney, as well as basic insights into the molecular nature of cell polarity.